There is a significant epidemiologic association between both ferritin-bound stored iron and dietary iron with an increased risk of breast cancer. Iron has been implicated in the initiation and progression of cancer through the generation of mutagenic reactive oxygen and as a component of ribonucleotide reductase, the rate-limiting enzyme in DNA synthesis. For these reasons, iron has emerged as a potential drug target in cancer and the use of iron chelators is currently being evaluated in clinical trials. Nitric oxide (NO) is upregulated in a variety of tumor types, including breast, and is often considered a negative prognostic indicator. This proposal is based on the discovery that when cancer cells are exposed to NO it reacts with free iron in the labile iron pool (LIP). This results in the quantitative conversion of this iron into paramagnetic dinitrosyliron complexes (DNIC). This proposal is aimed at testing the hypothesis that NO-mediated formation of DNIC serves two vital cellular functions: (1) short-term protection against iron-driven oxidative stress and (2) long-term inhibition of cell growth and migration by restricting the bioavailability of labile iron. Although much has been learned pertaining to the chemistry of DNIC assembly and degradation under synthetic laboratory conditions, there is a knowledge gap regarding the biological parameters that favor DNIC metabolism and the consequences of these reactions on tumor cell phenotype. We propose the following aims using breast cancer cells and NO-producing macrophages: Aim 1 is to test the hypothesis that labile iron is a principal target for NO in cancer cells. In Aim 2 we will demonstrate that the cytoprotective properties of NO are a consequence of DNIC formation. Finally, Aim 3 will test the hypothesis that NO suppresses tumor cell migration via iron sequestration. These aims are supported by our preliminary data which demonstrate that breast cancer cells treated with metal chelators recapitulate specific effects of DNIC formation. Methods: Using electron paramagnetic resonance (EPR) imaging we will quantify the amount and measure the kinetics of DNIC formation and disappearance in breast (MCF7, MCF10A, MDA-MB-231, HCC1806) and macrophage (RAW 264.7) cell lines following exogenous (NO-donors) and endogenous (NO producing macrophages) NO exposure. These results will demonstrate the cytoprotective effects of NO against damage induced by chemical oxidants and oxidant-generating chemotherapeutic agents. Lastly, using a label-free real-time cell migration assay we will measure the migratory ability of breast cancer cells in relation to iron in its labile or DNIC form. Significance: Iron is emerging as an important driving force for various types of cancers (including breast), leading to the use of iron chelators as potential treatments. Nitric oxide has also been implicated in various stages of cancer. This proposal will help solidify a new role for NO in cancer by demonstrating unique signaling and phenotypic responses attributable to DNIC formation. This has significance in regard to endogenous NO-producing tumors as well as NO-mediated chemotherapeutics. PUBLIC HEALTH RELEVANCE: Excess iron has been associated with an increased risk for the development of certain types of cancer, including breast. The free radical nitric oxide, which is produced by and around some tumors, can bind to iron rendering it unreactive. This proposal aims to study the interaction of nitric oxide with iron in hopes developing new chemotherapeutic strategies for a number of cancers.